Shigella sp. cause diarrhea and dysentery by invasion and spread through the colonic mucosa. Shigella sp. are CDC/NIAID priority pathogens. In the cell cytoplasm, Shigella move to the cell periphery and into adjacent cells by assembling propulsive actin tails. Within the cell body, assembly of actin tails involves the recruitment and activation of cellular N-WASP. In resting cells, N-WASP is maintained in a folded autoinhibited conformation. The molecular mechanisms by which intracellular Shigella activate N-WASP have previously been largely unknown. We have recently demonstrated that the cellular factor Toca-1 is required for S. flexneri activation of N-WASP, specifically for the relief of N-WASP autoinhibition. However, once actin tail assembly is initiated, Toca- 1 appears to no longer be required for ongoing polymerization of the actin tail. Our preliminary data indicate that actin tail assembly by S. flexneri is more efficient in the absence of the N-WASP regulatory protein WASP-interacting protein (WIP). N-WASP bound to S. flexneri has little WIP bound to it and migrates slower on SDS- PAGE. In addition, Toca-1 recruitment to the surface of intracellular bacteria depends on the bacterial type III secretion system, suggesting that a type III secreted protein is mediates Toca-1 recruitment. Our long-term goals are to understand the molecular mechanisms by which S. flexneri manipulate the cellular actin cytoskeletal machinery to enhance pathogenesis. In this R21 application, we propose exploratory studies on the mechanisms by which S. flexneri regulates the activation of N-WASP mediated actin assembly, with a focus on the role of WIP, Toca-1, and N-WASP modification on N-WASP activation. Our aims are to: 1. Characterize the role of WIP in S. flexneri activation of N-WASP. 2. Characterize S. flexneri modification of N-WASP and whether its modification leads to stabilization of the open active conformation. 3. Characterize the mechanism of Toca-1 recruitment to N-WASP on the surface of intracellular S. flexneri. Uncovering the mechanisms by which intracellular S. flexneri regulate the activation of cellular N-WASP is highly likely to generate important and novel insights both into the mechanisms of pathogenesis of S. flexneri and other bacterial pathogens and into the fundamental processes involved in activation and regulation of N-WASP in the cell. PUBLIC HEALTH RELEVANCE: The human pathogen Shigella is a bacterium that causes diarrhea by infecting cells that line the human intestinal tract and disseminating through intestinal tissue by mechanisms that are poorly understood. The bacterium promotes spread by producing molecules that enable the bacterium to use the scaffolding proteins of the cell to move through the tissue;we propose detailed studies into the molecular mechanisms enabling the bacterium to hijack the cell's scaffolding proteins in order to spread. Our results could lead to an improved understanding of how pathogens interact with human tissue and the development of better therapeutics.